Brain ischemia brings about hypoxic insults. Hypoxia is one of the major pathological factors inducing neuronal injury and central nervous system infection. We studied the involvement of mitogen-activated protein (MAP) kinase in hypoxia-induced apoptosis using cobalt chloride in C6 glioma cells. In vitro cytotoxicity of cobalt chloride was tested by MTT assay. Its IC 50 value was 400 µM. The DNA fragment became evident after incubation of the cells with 300 µM cobalt chloride for 24 h. We also evidenced nuclear cleavage with morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signal pathway of cobalt chloride-induced apoptosis in C6 cells. The activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) started to increase at 1 h and was activated further at 6 h after treatment of 400 M cobalt chloride. In addition, pretreatment of PD98059 inhibited cobalt chloride-induced apoptotic cell morphology in Electron Microscopy. These results suggest that cobalt chloride is able to induce the apoptotic activity in C6 glioma cells, and its apoptotic mechanism may be associated with signal transduction via MAP kinase (ERK 1/2).

Arsenic trioxide (As 2 O 3 ) was recently demonstrated to be an effective inducer of apoptosis in patients with relapsed acute promyelocytic leukemia (APL) as well as in patients with APL in whom all-trans-retinoic acid and conventional chemotherapy failed. Chronic myelogenous leukemia cells are highly resistant to chemotherapeutic drugs. To determine if As 2 O 3 might be useful for the treatment of chronic myelogenous leukemia, we examined the ability of As 2 O 3 to induce apoptosis in K562 cells. In vitro cytotoxicity of As 2 O 3 was evaluated in K562 cells by a MTT assay; the IC 50 value for As 2 O 3 was determined to be 10 µM. When analyzed by agarose gel electrophoresis, the DNA fragments became evident after incubation of the cells with 20 µM As 2 O 3 for 24 h. We also found morphological changes and chromatin condensation of the cells undergoing apoptosis. Activation of caspase-3 was observed 6 h after treatment with 20 µM As 2 O 3 by a Western blot analysis. Next, we examined the MAP kinasesignaling pathway of As 2 O 3 -induced apoptosis in K562 cells. As 2 O 3 at 10 µM strongly induced the activation of p38 and JNK 1/2, while ERK 1/2 was inhibited. In addition, pretreatment of SB203580, a specific inhibitor of p38, inhibited As 2 O 3 induced apoptotic cell death. These results suggest that As 2 O 3 is able to induce the apoptotic activity in K562 cells, and its apoptotic mechanism may be associated with the activation of p38.

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GDH has been known to be related with hyperinsulinismhyperammonemia syndrome. We have screened new drugs with a view to developing effective drugs modulating GDH activity. In the present work, we investigated the effects of a new drug, KHG26377 on glutamate formation and GDH activity in cultured rat islets. When KHG26377 was added to the culture medium for 24 h prior to kinetic analysis, the Vmax of GDH was decreased by 59% whereas Km is not significantly changed. The concentration of glutamate decreased by 50% and perfusion of islets with KHG26377 reduced insulin release by up to 55%. Our results show that KHG26377 regulates insulin release by inhibiting GDH activity in primary cultured islets and support the previous studies for the connection between GDH activity and insulin release. Further studies are required to determine in vivo effects and pharmacokinetics of the drug. [BMB reports 2010; 43(4): 245-249]

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